Fertility restorer gene and fertility restoration method for cw-type male sterile cytoplasm of rice

ABSTRACT

Mainly provided is a technique for directly identifying the genotype at locus Rf17 based on the specific base sequence data thereof. Also provided is a technique for artificially constructing a fertility-restored line. A method of restoring the fertility of CW-type cytoplasmic male sterile rice by inhibiting or reducing the expression of a gene comprising the base sequence represented by SEQ ID NO:2 in the above-described rice, and a method for determining the presence or absence of gene Rf17, which is a fertility restorer gene for CW-type cytoplasmic male sterility, comprising identifying a single nucleotide polymorphism (SNP) in the base at the 1812 position of the base sequence represented by SEQ ID NO:1 in the rice to be examined.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.12/922,071 filed Nov. 18, 2010, now allowed, which is a 371 U.S.National Stage application of PCT/JP2009/000753, filed Feb. 21, 2009,which claims priority from Japanese Application Serial No. 2008-062879,filed Mar. 12, 2008. The entirety of all the aforementioned applicationsare incorporated herein by reference.

The present invention relates to a method for restoring the fertility ofCW-type cytoplasmic male sterile rice, and to a method for determiningthe presence or absence of a fertility restorer gene and the like.Cytoplasmic Male Sterility (referred to hereinafter as “CMS”) means thatthe formation of pollen having the ability to germinate is inhibited dueto mutation in a mitochondrial gene so that a seed will develop intoseeds. It is known that fertility can be restored by the function of apollen fertility restorer gene (Rf) encoded in a nuclei in some cases ofCMS.

BACKGROUND

A method for breeding a first filial generation is also called a methodfor breeding of a hybrid-variety. This method is utilized invariety-breeding since a variety having excellent traits inherited fromits parents and showing heterosis hybrid vigour can be bred by means ofthis method. In order to economically harvest F1 hybrid seeds in largequantity, a three-line method utilizing cytoplasmic male sterility isadopted in the case of seed-harvesting of a hybrid variety of rice. Thethree-line method refers to a method that utilizes a sterile line havingmale sterility, a fertility restorer line, and a maintainer line havingthe same nuclear genes as the sterile line but not having sterilecytoplasm. According to this three-line method, (1) a hybrid seed can beharvested by crossing the sterile line with pollen of the restorer line,while (2) the sterile line can be maintained by crossing the sterileline with the pollen of the maintainer line.

BT-type male sterile cytoplasm and WA-type male sterile cytoplasm havebeen used worldwide in order to breed the hybrid variety with thethree-line method. On the other hand, CW-type male sterile cytoplasm hasbeen hardly_utilized since the structure and function of a fertilityrestorer gene Rf17 (Non Patent Document 1) have not been clarified.However, since there may be a risk of variety collapse due to limit inthe genetic resources provided by the male sterile cytoplasm that havebeen used until now, utilization and development of novel male sterilecytoplasm are now desired.

In the prior arts, it has been necessary to breed plant body (F1) from ahybrid seed obtained by test cross, and then to make the F1 self-crossand to investigate frequency of appearance of a individual plant havingseed-development rate ovevarietyrtain level (for example, 90% or more)for the estimation of a genotype of Rf17 locus in the plant. It has notbeen possible to determine said genotype by means of DNA markers.

-   Non Patent Document 1: Sota Fujii and Kinya Toriyama (2005)    Molecular mapping of the fertility restorer gene for ms-CW-type    cytoplasmic male sterility of rice. Theor. Appl. Genet. 111:696-701.

SUMMARY Problem to be Solved by the Invention

If the CW-type male sterile cytoplasm is utilized in the three-linemethod, it will be necessary to confirm that rice is holding Rf17 genein each step of breeding the rice of the restorer-line, and that itholds Rf17 gene in both alleles at a final stage. It is therefore a mainpurpose of this invention to provide a technique to directly identifythe genotype of Rf17 locus on the basis of its specific base sequence,and to provide a technique to artificially produce a fertility restorerline and the like.

Means for Solving the Problem

The present inventors have determined a base sequence comprising thefertility restorer gene Rf17 for the CW-type male sterile cytoplasm,which is represented by SEQ ID NO:1. We also have succeeded in therestoration of fertility of CW-type cytoplasmic male sterile rice byinhibiting the expression of a gene comprising the base sequencerepresented by SEQ ID NO:2, leading to the completion of the presentinvention.

Thus, the present invention relates to the following aspects:

1. A method for restoring the fertility of CW-type cytoplasmic malesterile rice, comprising inhibiting or reducing the expression of a genecomprising the base sequence represented by SEQ ID NO:2 in said rice.

2. The method according to claim 1, wherein the fertility is restored byintroducing a genomic fragment into the CW-type cytoplasmic male sterilerice, wherein the fragment is derived from the chromosome No. 4 of afertility restorer line for the CW-type cytoplasmic male sterility andcomprises nucleic acids consisting of the base sequence of at least atthe 1611-4835 positions of the base sequence represented by SEQ ID NO:1.

3. The method according to claim 2, wherein the genomic fragmentconsists of the whole base sequence represented by SEQ ID NO:1.

4. The method according to claim 1, wherein the fertility is restored byinhibiting the expression of the gene represented by SEQ ID NO:2 bymeans of RNA interference method.

5. The method according to claim 4, wherein the RNA interference isinduced by introducing a vector comprising a gene consisting of the basesequence represented by SEQ ID NO:2 or a continuous 100-500-basesequence in its 3′ non-translation region and their complementarysequence and expressing a double-stranded RNA that is capable ofinducing the RNA interference in a cell into the CW-type cytoplasmicmale sterile rice.

6. The method according to claim 5, which uses a vector comprising acontinuous base sequence at the 638-815 positions of the base sequencerepresented by SEQ ID NO:2 and its complementary sequence and expressinga double-stranded RNA that is capable of inducing the RNA interferencein a cell.

7. Rice whose fertility has been restored by the method according to anyone of claims 1-5.

8. A method for breeding a first filial generation variety using therice according to claim 7.

9. F1 hybrid seed harvested from the first filial generation varietybred by the method according to claim 8.

10. A method for determining the presence or absence of gene Rf17, whichis a fertility restorer gene for the CW-type cytoplasmic male sterility,comprising identifying a single nucleotide polymorphism (SNP) in a baseat the 1812 position of the base sequence represented by SEQ ID NO:1 inrice to be examined.

11. The method according to claim 10, wherein the SNP is identified bymeans of CAPS method.

12. The method according to claim 11, which uses a restriction enzymerecognizing the cleavage site GT(A)AAC.

13. The method according to Claim 12, wherein the restriction enzyme isMaeIII.

14. A kit used for the method according to any one of claims 10-13.

Advantages of the Invention

The present invention has revealed that the CW-type male sterility isrestored by the reduction of the expression of ORF11. And, the presentinventors have succeeded in the identification of a base sequence thatis effective in the restoration of the CW-type male sterility and in therestoration of said fertility.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A pattern diagrams showing a gene present in the gene fragmentsthat restored fertility in a complementarity test.

FIG. 2 Photos showing the presence or absence of decomposition of thestarch of pollen in a transformed plant into which a vector inducing RNAinterference has been introduced. The decomposition of starch wasobserved in the fertility restorer line (CWR) and an individual plant(RNAi ORF11_(—)3) into which an RNA interference vector for ORF11 hasbeen introduced, but it was not observed in the male sterile line (CWA)and an individual plant (RNAi PPR2_(—)1) into which an RNA interferencevector for PPR2 has been introduced.

FIG. 3 Photos of electrophoresis showing the determination of the Rf17genotype by means of PCR with a CAPS marker. While the fertilityrestorer line (CWR) generated a band of 370 bp, a variety having nofertility restorer capability (Taichung 65: T65) generated bands with276 bp and 84 bp.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors have succeeded in the determination of a genomicregion comprising the fertility restorer gene Rf17 for the CW-type malesterile cytoplasm in the chromosome No. 4 of rice, which is representedby SEQ ID NO:1, by means of a conventional map-base cloning method. Wealso have further succeeded in the restoration of fertility of theCW-type cytoplasmic male sterile rice by inhibiting or reducing theexpression of a gene (ORF11) represented by SEQ ID NO:2. The function ofthe gene represented by SEQ ID NO:2 has not yet known.

The map-base cloning method is also called a “chromosome walking”, whichfocuses a genomic region in which a gene to be isolated exists by meansof a DNA marker that is present in the vicinity of said gene. Thismethod is used as one of isolation methods of a gene when the functionor translation product of the gene can not estimated. Although thismethod has been utilized for the gene-isolation in some kinds of animalsand plants, it is more effectively used in the plants from which asegregating population for laboratory use may be produced according to aplan. The genomic sequence of nipponbare-type rice(http://rgp.dna.affrc.go.jp/J/index.html) was utilized in the map-basecloning according to the present invention.

The present inventors have found a way of introducing a genomic fragmentinto the CW-type cytoplasmic male sterile rice, wherein the fragment isderived from the chromosome No. 4 of a fertility restorer line for theCW-type cytoplasmic male sterility and comprises nucleic acidsconsisting of the base sequence of at least at the 1611-4835 positionsof the base sequence represented by SEQ ID NO:1 in order to inhibit orreduce the expression of the gene comprising the base sequencerepresented by SEQ ID NO:2. The whole base sequence represented by SEQID NO:1 is listed as one of the examples of the above genomic fragments.

The examples of the above genomic fragment further include a nucleicacid which may hybridize with a nucleic acid consisting of the basesequence complementary with the base sequence represented by SEQ ID NO:1or its part under stringent conditions, and a nucleic acid consisting ofthe base sequence having homology of about 80% or more, preferably about95% or more with the above nucleic acids. Those nucleic acids canrestore the fertility of CW-type cytoplasmic male sterile rice.

The hybridization may be carried out by or according to a method knownin the art such as, for example, Molecular cloning third. ed. (ColdSpring Harbor Lab. Press, 2001). Commercially available libraries may beused in accordance with the description of an attached instruction foruse.

The “stringent conditions” in the present specification means, forexample, the temperature of 60-68° C., sodium concentration of 150-900mM, preferably 600-900 mM and pH6-8.

Accordingly, the above nucleic acid which may hybridize with a nucleicacid consisting of the base sequence complementary with the basesequence represented by EQ ID NO:1 or its part may be a nucleic acidcomprising the base sequence having homology of about 80% or more,preferably about 95% or more, more preferably 99% or more with the wholebase sequence of said nucleic acids.

The sequences may be pre-treated into a suitable state for comparisonbefore homology (identity) is determined between two base- or aminoacid-sequences. For example, a gap may be introduced into the sequenceof one of them so as to optimize alignment with the other sequence. Theamino acid or base sequences will be compared in each part thereof. Whena part of the first sequence has the same amino acid or base sequence asa corresponding part of the second sequence, their sequences are deemedto be identical with each other in those parts. Homology between the twosequences is shown as a percentage of the number of the same amino acidsor bases for the total number of the amino acids or bases in that part.

According to the above principle, homology (sequence homology) betweentwo base sequences may be determined by means of algorism of Karlin andAltschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990 and Proc. Natl.Acad. Sci. USA 90:5873-5877, 1993). BLAST and FAST programs based on theabove algorism are used to investigate a sequence having a high homologywith a given sequence from data base. These programs are available inthe website of the National Center for Biotechnology Information on theInternet.

The nucleic acid having the above homology in the base sequence may beobtained using hybridization as an index, or obtained from a group ofunidentified DNAs that have been obtained by the analysis of genomicbase sequences or from public database by using any method that thoseskilled in the art usually uses, such as BLAST software. The geneaccording to the present invention may also be obtained by a knownmutation-introducing method.

The expression of the gene comprising the base sequence represented bySEQ ID NO:2 in the CW-type cytoplasmic male sterile rice may beinhibited or reduced by any other methods known for those skilled in theart. For example, the fertility of the CW-type cytoplasmic male sterilerice may be restored with anti-sense RNA method or by the inhibition ofthe expression of the gene represented by SEQ ID NO:2 with RNAinterference method in accordance with the Example of the presentspecification.

Thus, the RNA interference may be induced by introducing a vector intothe CW-type cytoplasmic male sterile rice, wherein the vector isdesigned to express a double-stranded RNA capable of inducing the RNAinterference so as to inhibit or reduce the expression of the genecomprising the base sequence represented by SEQ ID NO:2. The vectorincludes one comprising a gene consisting of the base sequencerepresented by SEQ II) NO:2 or a continuous 100-500-base sequence,preferably a continuous 150-200-base sequence in its 3″ non-translationregion and their complementary sequence and expressing a double-strandedRNA that is capable of inducing the RNA interference in the cell. Anexample of said vector is one comprising a continuous 178-base sequenceat the 638-815 positions of the base sequence represented by SEQ ID NO:2(its first base is numbered as “1 position”) and its complementarysequence and expressing a double-stranded RNA that is capable ofinducing the RNA interference.

The above vector may be prepared by combing the above DNA within avector according to any suitable gene-engineering technique known forthose skilled in the art. The above vector may comprise a suitablepromoter and any other controlling sequences, for example, enhancersequence, terminator sequence, polyadenylation sequence and the like inorder to express the gene according to the present invention in a hostcell.

The genomic region or the vector to be used for the inhibition orreduction of the expression of the gene comprising the base sequencerepresented by SEQ ID NO:2 in the CW-type cytoplasmic male sterile ricemay be introduced into a subject rice by any method known for thoseskilled in the art such as Agrobacterium method, free-thaw method andelectroporation method.

As a result, the fertility is restored in the CW-type cytoplasmic malesterile rice. Accordingly, a first filial generation variety is bredusing the rice whose fertility has thus been restored so that an F1hybrid seed can be harvested from the variety.

The present invention is further relates to a method for determining thepresence or absence of a fertility restorer gene for the CW-typecytoplasmic male sterility, Rf17 or a genomic region comprising thegene, comprising identifying a single nucleotide polymorphism (SNP:A/T)in a base at the 1812 position of the base sequence represented by SEQID NO:1 in the rice to be examined.

The determination of SNP may be carried out by any method known forthose skilled in the art, such as base sequence-determination method,SSCP (single strand conformation polymorphism) method, ASA (Allelespecific amplification), primer extension method, Taqman method,invasion method, dot-blot-SNP Method, FRIP (Fluorogenic RibonucleaseProtection) method, and TILLING (Targeting Induced Local Lesion inGenome) method.

There is also CAPS (Cleaved Amplified Polymorphic Sequence) method forthe above determination, as described in the Example of the presentspecification. In this method, a primer is so designed that arestriction enzyme-recognition site will be formed at a location wherethe SNP exists, followed by PCR-RFLP analysis on polyacrylamide gel.This method has an advantage that its results will hardly be affected bythe conditions of an experimenter or a sample, or the kind of a DNAextraction method so as to easily obtain constant results. Furthermore,the method can be performed in a relatively simple way and does not needan expensive apparatus such as a DNA sequencer or advanced technique.

Primers, markers or probes that are used in the above method for theidentification of SNP may be easily designed and prepared by thoseskilled in the art in accordance with the principles of each method onthe basis of the database described above and the information about theDNA sequence of SEQ ID:NO1 or NO:2 disclosed in the presentspecification. For example, a primer used in PCR of the CAPS methodusually has the length of several tens of by such as 10-30 bp.

Various kinds of oligonucleotides that will be used as the aboveprimers, markers or probes may be synthesized in vitro by any methodknown for those skilled in the art, such as chemical synthesis methodsthat are described, for example, in Carruthers (1982) Cold Spring HarborSymp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661;Belousov (1997) Nucleic Acid Res. 25:3440-3444; Frenkel (1995) FreeRadic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896;Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066. Any knownlabeling substance may be attached to them for detection.

As a sample of rice to be examined, any part of the rice may be usedsuch as its seed, leaf and stem. DNA may be extracted and prepared byany method known for those skilled in the art from the above sample. Thekind of DNA to be prepared from the sample has no limitation, a genomicDNA (gDNA) and cDNA being listed for example. They may be extracted andpurified by any method known for those skilled in the art depending ontheir properties, and kinds and properties of the sample. For example,gDNA may be obtained by CTAB method, boiling method and enzyme methodusing amylase or protease, if necessary.

If a sufficient amount the DNA is extracted from the sample rice fordetection, it will be subjected to the following procedures withoutamplification. However, the DNA will be usually amplified to an amountsuitable for the identification of SNP by means of anygene-amplification method known for those skilled in the art, such asPCR (Polymerase Chain Reaction) or RT-PCR method, ICAN (Isothermal andchimeric primer-initiated amplification of nucleic acids) method, NASBA(Nucleic acid sequence based amplification) method, TMA(Transcription-mediated amplification) method and SDA (StrandDisplacement Amplification) method.

The present invention also relates to a kit used for the method fordetermining the presence or absence of gene Rf17. Depending on the kindsof SNP analysis and the like, the kit comprises primers, markers orprobes for the identification of SNP. It may further optionally comprisevarious primer set and/or marker for the amplification of DNA, arestriction enzyme and other elements or components known for thoseskilled in the art such as, for example, various agents, enzymes, andbuffer, a reaction plate (vessel) depending on its structure andpurpose.

Although the present invention will be explained in more detail withreference to the following Examples, the technical scope of the presentinvention shall not be construed to be limited by them. The termsdescribed in the present specification are used in such meaning as isusually used in the art unless particularly noted otherwise.

Unless otherwise described, each procedure may be carried out inaccordance with standard techniques in gene engineering and molecularbiology known for those skilled in the art, such as those described inSambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, ColdSpring Harbor Laboratory Press, New York, 1989; Molecular cloningthird.ed. (Cold Spring Harbor Lab. Press, 2001); Ausubel, F. M. et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., 199 and the like. The content of the publications cited in thepresent specification will constitute a part of the disclosure of thepresent specification.

Example 1 Restoration of the Fertility by Introduction of a GenomicFragment of the Base Sequence Represented by SEQ ID NO:1

Mapping of Rfl1 using the fertility restorer line (CWR line) for theCW-type CMS line revealed that Rf17 exists in a region of 77 kb of thechromosome No. 4, so that the base sequence of that region wasdetermined. The genomic region was divided into seven fragments andsubjected to sub-cloning. Each genomic fragment was then introduced intothe CW-type CMS line by means of Agrobacterium method and the seedfertility of the transformed plant was investigated. A plant individualwhose fertility had restored was obtained from the plant transformedwith the genomic fragment No. 5 (Table.1). Fertility was restored infour (4) individuals among 44 re-differentiation lines. The fourindividuals showed the seed fertility of 79.3, 23.1, 25.3 and 68.1%,respectively. The base sequence of the fragment No. 5 is shown as SEQ IDNO:1.

TABLE 1 No. of Individuals of No. of Individuals of No. of GeneIntroduction Re-differentiation Fertility Restoration 1 17 0 2 9 0 3 8 04 10 0 5 44 4 6 7 0 7 6 0

Two genes were predicted in the above fragment, which were PPR gene anda functionally unidentified gene (named as “ORF11”) (FIG. 1). Thecomparison in expression between the CMS line and the fertility restoredline revealed that the expression of ORF 11 was high in the CMS line butlow in the fertility-restored line, while there is no difference in theexpression of PPR gene between them. The reduction of the expression ofORF 11 was also observed in the next generation of the transformationline whose fertility had been restored. The comparison of base sequencebetween the CMS line and the fertility-restored line showed that therewas a single base-substitution within the PPR gene, which generated astop codon in the fertility-restored line (FIG. 1). As the singlebase-substitution was located 5′-upstream of ORF11, it was consideredthat the expression of ORF11 was reduced in the fertility-restored linedue to the above mutation. It was therefore concluded that the fertilitywas restored due to the reduction of the expression of ORF 11.Accordingly, it has been revealed that the fertility is restored byintroducing a genomic fragment comprising the base sequence representedby SEQ ID NO:1, into the CW-type cytoplasmic male sterile line.

The “PPR” is an abbreviation for “pentatricopeptide repeat” that isprotein having a repeated conserved sequence consisting of 35 aminoacids. The protein is considered to bond to the RNA of arganelle and beinvolved in RNA processing and regulation of translation.

Example 2 Restoration of the Fertility by of the Gene of SEQ ID NO:2

Mapping of the fertility restorer gene, Rf17, for the CW-type CMS linerevealed 14 candidate genes within the candidate region of 77 kb.According to polymorphism analysis of the CMS line and the fertilityrestorer (CWR) line, an amino acid mutation had occurred only in the PPRgene (“PPR2 gene” in FIG. 1) and the stop codon was generated in anallele of the CWR line. According to expression analysis done for eachof the 14 candidate genes, only ORF 11 gene showed the differencebetween the CMS line and the CWR line. The base sequence comprising acoding region, and its 5′- and 3′-non translation regions are shown asSEQ ID NO:2. A base sequence corresponding to that at the 638-815positions of the base sequence represented by SEQ ID NO:2 (its firstbase is numbered as “1 position”) and a base sequence corresponding tothat at the 772-1505 positions of the base sequence represented by SEQID NO:1 (its first base is numbered as “1 position”), which correspondsto PPR2 gene, were amplified by means of PCR using the primersrepresented by SEQ ID NO:3 and NO:4, and the primers represented by SEQID NO:5 and NO:6, respectively, and linked down stream of a ubiquinonepromoter of pANDA vector to give a vector that induces the RNAinterference. The resulting vectors were introduced into the CW-type CMSline by means of Agrobacterium method, and shape of pollen and seedfertility were investigated. As a result, the expression of ORF11 wasreduced down to 30-77% in seven (7) lines into which the RNAinterference-inducing vector for ORF11 had been introduced. And, four(4) lines out of said seven lines showed decomposition of the starch ofpollen, which is characteristic to the fertility restorer line (FIG. 2),and their seed fertility was partially restored (2-3%). On the otherhand, although the expression of ORF11 was reduced down to 27-75% in six(6) lines into which the RNA interference-inducing vector for PPR2 hadbeen introduced, no decomposition of the starch of pollen was observedlike the CMS line (FIG. 2) showing the seed fertility of 0%. The aboveresults demonstrated that the fertility of the CW-type CMS line can berestored by inhibiting or reducing the expression of the gene ORF 11.

Example 3 Determination of Rf17 Genotype

Comparison of the base sequences represented by SEQ ID NO:1 comprisingthe fertility restorer gene Rf17 with that of Nipponbare-type ricerevealed that a base at the 1812 position of the base sequencerepresented by SEQ ID NO:1 is “T” in the fertility restorer (CWR) line,while “A” in the nipponbare-type rice that has no fertility-restoringcapacity. A CAPS marker was prepared for a simple identification of thismutation. PCR was carried out using two primers represented by SEQ IDNO:7 and NO:8. Treatment with a restriction enzyme MaeIII andelectrophoresis gave a band with 370 bp in the fertility restorer line(CWR), and bands with 276 bp and 84 bp in the nipponbare-type rice (FIG.3). The above results showed that the present or absence of Rf17 couldbe simply determined. By the way, the restriction enzyme that can beused in the above determination is not limited to MaeIII, but anyrestriction enzyme may be used as long as it recognizes the cleavagesite GT(A)AAC.

INDUSTRIAL APPLICABILITY

According to the present invention, a hybrid variety can be bred by thethree-line method using the CW-type male sterile cytoplasm.

1-14. (canceled)
 15. A polynucleotide consisting of a base sequenceselected from the group consisting of: (a) a base sequence at least1611-4835 positions of the base sequence represented by SEQ ID NO:1; (b)a base sequence that hybridizes with the base sequence (a) or itscomplementary sequence under stringent conditions, and (c) a basesequence having homology of about 95% or more with the base sequence(a).
 16. A vector comprising the polynucleotide according to claim
 1517. A plant that is transformed with the vector according to claim 16.18. The plant according to claim 17, said plant is transformed by meansof Agrobacterium method.
 19. A plant of CW-type cytoplasmic male sterilerice, said plant is transformed with the vector according to claim 16,so that its fertility has been restored.
 20. The plant according toclaim 19, said plant is transformed by means of Agrobacterium method.21. A polynucleotide consisting of contiguous 10-30 bp comprising a base“T” at position 1812 of the base sequence represented by SEQ ID NO:1.22. A primer, marker or probe comprising the polynucleotide according toclaim 21 for use in the identification of a single nucleotidepolymorphism in a base at 1812 position of the base sequence representedby SEQ NO:1.